The present invention relates to methods for determining the immunological status of transplant recipients (and kits used for such purposes). More particularly, it relates to non-invasive techniques for discriminating between non-rejecting human organ transplant recipients and those experiencing rejection.
Organ transplantation is an important therapeutic option for patients with end-stage organ diseases. Although improved immunosuppressive strategies have dramatically increased the rate of short-term success of such transplants, the rate of long-term transplantation success has not improved as significantly, primarily due to a high incidence of rejection episodes. Rejection episodes, if not quickly diagnosed and treated, often significantly and adversely affect long-term function of the transplanted organs.
Rejection can be detected by observing gross symptoms (e.g. elevated temperature; significant failures of the function of the transplanted organs). However, if one waits for gross symptoms to appear before modifying immunosuppressive therapy, long-term injury may have already occurred to the transplanted organ. Thus, bioassays have been developed to try to detect early stages of rejection episodes. Various bioassays have been developed. See generally U.S. Pat. Nos. 4,959,302; 5,223,396; 5,334,504; and 5,482,841. The disclosure of these publications, and all other publications referred to herein, are incorporated by reference as if fully set forth herein.
Such bioassays are also desirable to render more efficient immunotherapy. In this regard, when doctors have confidence in their ability to spot the early onset of rejection episodes, they are more willing to try to minimize use of immunotherapeutic drugs (and thereby reduce the costs of such drug treatment and the risk of drug-associated side effects).
One existing bioassay approach is to measure general T cell activation by (i) spontaneous blastogenesis in which freshly isolated lymphocytes from the transplant recipient are examined from time to time for [.sup.3 H] thymidine uptake, and (ii) expression of surface molecules associated with T cell activation such as the receptor CD25. A drawback of this approach is the incidence of false positive results (as such assays do not discriminate well between T cell activation due to rejection and that due to viral or bacterial infection).
False positives can lead to the patient having to undergo unneeded prophylactic drug therapy, as well as unnecessary emotional stress.
Another bioassay approach assesses activation status of donor-specific T cells isolated from graft biopsies. This determination is by the mixed lymphocyte reaction in which lymphocytes isolated from the graft are examined for [.sup.3 H] thymidine uptake after incubation with irradiated donor lymphoid cells. A variant is a determination by cell-mediated lympholysis in which lymphocytes isolated from the graft are examined for their ability to lyse chromium-labeled donor lymphoblastoid cells. Graft-derived endothelial and epithelial cells have also been used to stimulate recipient lymphocytes isolated from the graft.
Major drawbacks with respect to these assays are that these assays typically use growth factors to propagate and potentiate the responsiveness of recipient T cells, a process that alters T cell responsiveness. This is important because the presence of donor-specific lymphocytes in the organ is not by itself an indicator of rejection, since cellular infiltrates can often be detected in the graft without any signs of pathological changes associated with rejection.
Also, the donor lymphocytes commonly used in these assays to stimulate donor-specific recipient T cells may not bear graft-specific antigens, leading to false negative results. Moreover, as with histological and gene expression analysis, such graft-specific bioassays also require graft biopsies. As a result of these deficiencies, the biological assays that have been commercialized for monitoring transplanted organ rejection status (prior to gross rejection symptoms appearing) have not gained widespread acceptance.
The most dependable tool currently used for diagnosing graft rejection is biopsy histology. However, repetitive biopsy for purposes of monitoring transplant status can be invasive and expensive. Also, this technique can sometimes be too insensitive to detect early, subtle cytopathic processes. Further, reading of histological slides for this purpose requires special expertise to accurately and consistently interpret results, thus raising its cost as well as the risk of occasional improper interpretation.
As such, it can be seen that the need exists for improved bioassay techniques for monitoring the immunological status of human transplant recipients.